CN111118200B - CAPS marking method for distinguishing banana wilt resistant varieties - Google Patents
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Abstract
The invention provides a CAPS marking method for distinguishing fusarium wilt resistant varieties of bananas, which can be used for distinguishing the InDel mutation of the medium heat 1 and Brazilian bananas by sequencing and analyzing the medium heat 1 and the Brazilian bananas, wherein the mutation causes the difference of gene sequences on BclI endonuclease digestion.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a CAPS (CAPS marker system) marking method for distinguishing banana vascular wilt resistant varieties.
Background
The restriction amplified polymorphic sequence (CAPS) is a molecular marking method generated by combining PCR reaction and restriction endonuclease digestion. The basic principle is that a set of specific PCR primers is designed by using DNA sequences of known sites, and after amplification, amplified products obtained by enzyme digestion of a specific restriction endonuclease are used for restriction fragment length polymorphism analysis. The CAPS marker has the characteristics of co-dominance, specificity, simple operation and low cost, and is mainly used for researches on aspects such as plant gene localization, parting, map-location cloning, variety and strain identification and the like at present.
Bananas are an important tropical commercial crop with both fruits and grains. Bananas are currently grown in more than 130 countries around the world. The planting area in China is 41.28 million hectares (without Taiwan), the yield reaches 1085 million tons, the yield value is $ 29.7 hundred million, and the second place is in the world (FAOSTAT, 2014). The banana industry has become the backbone industry of tropical agriculture in China, and plays an important role in southern hotspot economy and rural social development. Currently, the outstanding problems facing the banana industry are mainly: firstly, the main cultivated species is single, and the fine species is lack. No matter in China or other banana producing countries, the main cultivated varieties of the banana plants are mainly Brazilian and Williams of the Cavindish type, and the character of the banana plants is degraded due to long-term single cultivation and asexual propagation. The banana industry has been stricken destructively by various pests and diseases in each banana producing area for a long time, especially banana wilt. Secondly, the breeding means is deficient, and the breeding of new species is difficult. The main cultivar of banana is triploid, the breeding mode is difficult to adopt cross breeding, the breeding mode of banana mainly induced by radiation needs to keep a large number of screening groups, and excellent plant screening is mainly marked by form, so that the breeding has long screening period and high cost of manpower, land and the like.
Therefore, there is a strong need for good varieties and development of effective molecular markers or means for early selection of mutagenized progeny in banana production. The genetic mechanism of the banana excellent character related gene is analyzed from the gene level, and the molecular marker for early screening and identification is developed, so that the method has important guiding and practical significance for auxiliary selection of banana molecular breeding and variety breeding.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a CAPS marking method for distinguishing banana wilt resistant varieties based on InDel marks of Banana (Banana) Zhonghong No. 1 and Brazil bananas, can effectively identify Banana (a main cultivar) and Banana Zhonghong No. 1 in a disease-resistant variety bred by radiation mutation breeding, can be applied to identification of banana resistant varieties, and lays a foundation for banana resistance mutation breeding.
Zhonghong No. 1 is obtained by using Brazil banana60The new variety of banana with blight resistance obtained by Co gamma ray radiation mutagenesis and directed breeding has been reported in 2017, 04 and 05.s.t. application No. 20172338.6.
The invention provides a CAPS marking method for distinguishing banana vascular wilt resistant varieties, which comprises the following steps:
(1) extracting the DNA of the banana genome to be detected;
(2) taking the genome DNA of the banana to be detected as a template, and carrying out PCR amplification reaction by using a specific primer pair, wherein the specific primer pair comprises:
forward primer G0629F 2: 5'-TAGCAAAATACATGAACTTTTGGA-3' the flow of the air in the air conditioner,
reverse primer G0629R 2: 5'-TCCTCACGACAACAACGAGAAAAAG-3', respectively;
(3) and carrying out enzyme digestion on the amplification product by using BclI restriction enzyme to obtain an enzyme digestion product, carrying out gel electrophoresis on the enzyme digestion product, wherein if two fragments are obtained, the obtained product is Brazil banana, and if three fragments are obtained, the obtained product is Zhonghui No. 1 banana.
In the step (3), if two fragments with the sizes of 447bp and 196bp are obtained, the banana is Brazil banana, and if three fragments with the sizes of 447bp, 129bp and 77bp are obtained, the banana is Zhongzhou No. 1 banana.
The second aspect of the invention provides a primer pair for a CAPS marking method, wherein the primer pair comprises: forward primer G0629F 2: 5'-TAGCAAAATACATGAACTTTTGGA-3', reverse primer G0629R 2: 5'-TCCTCACGACAACAACGAGAAAAAG-3' are provided.
The third aspect of the invention provides an InDel molecular marker for distinguishing banana vascular wilt resistant varieties, wherein the InDel molecular marker is positioned at the 128bp position of a banana calcium-dependent protein kinase gene promoter, the sequence of the InDel molecular marker is CATCATTCTTG, the insertion of the banana with the number 1 of Zhongzhonghong is CATCATTCTTG, and the deletion of the banana with the number CATCATTCTTG.
The fourth aspect of the invention provides the application of the InDel molecular marker in the third aspect of the invention in distinguishing banana vascular wilt resistant varieties.
The fifth aspect of the invention provides application of a primer pair capable of identifying the InDel molecular marker in the third aspect of the invention in preparing a reagent for distinguishing banana vascular wilt resistant varieties.
According to the invention, through sequencing and analyzing the Zhongzhong No. 1 banana and the Brazil banana, InDel mutation for distinguishing the Zhongzhong No. 1 banana from the Brazil banana is obtained, and the mutation causes the difference of the gene sequence on Bcl I incision enzyme digestion.
Drawings
FIG. 1 is a diagram of banana calcium dependent protein kinase gene promoter region sequence amplification and mutation site analysis.
FIG. 2 shows CAPS analysis of the mutant site of the promoter region of banana calcium-dependent protein kinase gene and analysis of the site of amplification sequence endonuclease: a is CAPS analysis of the mutant site of the promoter region; and B is enzyme cutting site analysis of the promoter region amplification sequence.
FIG. 3 is the PCR amplification and digestion map of banana calcium dependent protein kinase gene promoter: a is Brazil and moderate heat No. 1 PCR amplification electrophoresis picture, B is Brazil and moderate heat No. 1 PCR product enzyme cutting banding pattern picture, wherein, M is DL2000 marker, 1-3 is Brazil banana PCR amplification and enzyme cutting sample; 4-6 are the samples of the heat No. 1 PCR amplification and enzyme digestion.
Detailed Description
The invention will be better understood from the following description of specific embodiments with reference to the accompanying drawings. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
1. Genomic DNA extraction
Selecting 4 Brazilian banana healthy plants and the Zhongzhou No. 1 banana healthy plants respectively at random, taking tender reel leaves as materials, extracting genome DNA by utilizing a polysaccharide polyphenol plant genome DNA extraction kit (DP360) of Beijing Tiangen Biochemical technology Co., Ltd, and detecting the DNA quality by 1% agarose gel electrophoresis.
2. Identification of SNP and InDel mutation sites
The results of the re-sequencing and transcriptome sequencing of the heat 1 and Brazilian banana in the early-stage infection-resistant variety in the laboratory show that the expression of a resistance-related gene, namely a calcium-dependent protein kinase gene (CDPK, Ma04t06290.1), in 2 banana varieties has obvious difference, and the gene is possibly related to the resistance of the blight. The sequence of the promoter region of the gene was extracted, primer pairs (G0629F 1: 5'-TAGCAAAATACATGAACTTTTGGAG-3' and G0629R 1: 5'-GGTCTTTTACCAACCAAAAATAAGC-3') were designed, and the genomic DNA of Zhonghong No. 1 and Brazil bananas was subjected to PCR amplification.
The PCR amplification system contained 1.0. mu.l of DNA template, 2.0. mu.l of 10 XEx Taq Buffer, 1.0. mu.L of TaKaRa Ex Taq, 1.0. mu.l of each of 10. mu.M upstream and downstream primers, and 14.0. mu. L H2O. The reaction program is pre-denatured at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 5 min.
And (2) recovering and purifying the PCR product by using 1% agarose gel electrophoresis, recovering the fragment and connecting the recovered fragment with a pMD19-T vector, transforming escherichia coli DH5 alpha, selecting positive clone sequencing, wherein the sequence of the promoter region of the Brazilian banana calcium-dependent protein kinase gene is shown as SEQ ID NO. 1, and the sequence of the promoter region of the moderate heat No. 1 calcium-dependent protein kinase gene is shown as SEQ ID NO. 2. And (3) carrying out sequence comparison on the sequence obtained by sequencing by using DNAMAN software, and identifying SNP and InDel sites between two varieties with different anti-infection properties. Found by sequence alignment (figure 1): a clear single sequence of about 1130bp was amplified in both varieties, and there were 1 InDel site (CATCATTCTTG) 11bp long at 128bp of the amplified sequence, while there were also 10 base mutations in this region, at 147, 155, 185, 277, 291, 315, 317, 359, 428 and 726bp sites of the sequence, respectively.
3. Development of CAPS markers
Bioinformatics analysis of the variant sites present in these two varieties revealed that the InDel insertion mutation at position 128 produced a Bcl I restriction endonuclease site (T ^ GATCA) in the resistant varieties. Therefore, CAPS primers (G0629F 2: 5'-TAGCAAAATACATGAACTTTTGGA-3' and G0629R 2: 5'-TCCTCACGACAACAACGAGAAAAAG-3') were designed on both sides of the region containing the InDel mutation site, and the sizes of the product fragments after PCR amplification and enzyme cleavage by using the primer sequences are shown in Table 1. The 25.0. mu.l enzyme digestion reaction system comprises: 5.0. mu.l of PCR product, 2.5. mu.l of 10 Xdigestion buffer, 0.5. mu.l of endonuclease, and 17.0. mu.l of water. The reaction was carried out at 37 ℃ for 1 h. The cleavage products were detected by 2.0% agarose gel electrophoresis.
As shown in FIG. 2A, 643bp and 654bp fragments can be amplified in Brazil banana and Zhonghong No. 1 varieties, and sequencing analysis is performed on the amplified sequence to find that: this sequence still contained 1 BclI cleavage site in the non-variant region except that the site of variation contained 1 BclI cleavage site, so that the Zhonghui No. 1 contained 2 BclI enzymatic cleavages in the sequence fragment and the Brazil banana was 1 Bcll enzymatic cleavage (FIG. 2B). Thus, by performing the amplification and enzyme digestion electrophoresis on Brazil banana and Zhonghuan No. 1, two fragments with sizes of 447bp and 196bp, respectively, can be obtained in Brazil banana, and three fragments with sizes of 447bp, 129bp and 77bp, respectively, can be obtained in Zhonghuan No. 1 (FIG. 3).
TABLE 1G 0629PCR products and restriction enzyme products
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Sequence listing
<110> research institute of tropical biotechnology of Chinese tropical academy of agricultural sciences
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tagcaaaata catgaacttt tggagaacaa cattaatttt aaaactgaga taaatgatta 60
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tcgtgattat tgattagttt ttataaaaaa aaatcaaact aaattaagat ttttattttt 180
tcatcaaaat tgatcaaaca taattccatc aagtcggaca caatcgatgt ctcacgagtc 240
caattgccat cggtcacttt ccgtctttca ctgtcattag taatattgca tgtgacgtat 300
aatgtacagt gaacgatcaa atacgacgtt ccgatttcga ttagaatata agaactatat 360
tttgagtagg aagtcgtcaa cacatccctt ctgcttaaat caatacggta cttctcttgt 420
cccgctgaag tcgcgtatga atcggtcaag tctggcgtcc aaatgcatct tggagcgcag 480
gcgaagaggg acacgttggt catcacgaac tccgttagat ttccgacgag cgaggagaaa 540
cgatcgcgtg tttcttgtga tgctgttttg actattacaa cccaagaaag aaggcggttt 600
ttgcttctcg tttggcttct ttttctcgtt gttgtcgtga ggagctggtg ggagggaggg 660
gagaagagag tcggaagctt tgacgctctc ttctcacgac attgggctca ttcgaccctc 720
ctgctcttga ttggaattcg aaatcttttg ttccaatccg tttgttttct tctcgcgcgg 780
accctttgtt tgacttctta cgtcgtaaaa attcgtactt tgccgctgtt gccgttcgag 840
ttgagagaat ccgagagggg aggagataac agaggggtgc ggaagctcgg attctcgttt 900
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tcgtgatcat cattcttgta tagattaatt tttattaaaa aaaatcaaac taaattaaga 180
tttttgtttt ttcatcaaat tgatcaaaca taattccatt aagtcggaca caatcgatgt 240
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cttctctcgt cccgctgaag tcgcgtatga atcggtcaag tctggcgtac aaatgcatct 480
tggagcgcag gcgaagaggg acacgttggt catcacgaac tccgttagat ttccgacgag 540
cgaggagaaa cgatcgcgtg tttcttgtga tgctgttttg actattacaa cccaagaaag 600
aaggcggatt ttgcttctcg tttggcttct ttttctcgtt gttgtcgtga ggagctggtg 660
ggagggaggg gagaagagag tcggaagctt tgaccctctc ttctcacgac attgggctca 720
ttcgatcctc ctgctcttga ttggaattcg aaatcttttg ttccaatccg tttgttttct 780
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gccgttcgag ttgagagaat ccgagagggg aggagacagc agaggggtgc ggaagctcgg 900
attctcgttt ttggaagaag aacggaggaa agaagcaggc gaattgagac aaggaaagaa 960
aaacacaagg gttcctgttc tcgtttctct tgtcttcgtt cctttcaatt cgaggagttc 1020
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Claims (6)
1. A CAPS marking method for distinguishing Brazil bananas and Banana Zhonghui No. 1 is characterized by comprising the following steps:
(1) extracting banana genome DNA to be detected, wherein the banana genome DNA to be detected is derived from the Banana heat No. 1 or Brazil banana;
(2) taking the genome DNA of the banana to be detected as a template, and carrying out PCR amplification reaction by using a specific primer pair, wherein the specific primer pair comprises:
forward primer G0629F 2: 5'-TAGCAAAATACATGAACTTTTGGA-3' the flow of the air in the air conditioner,
reverse primer G0629R 2: 5'-TCCTCACGACAACAACGAGAAAAAG-3', respectively;
(3) and carrying out enzyme digestion on the amplification product by using Bcll restriction enzyme to obtain an enzyme digestion product, carrying out gel electrophoresis on the enzyme digestion product, wherein if two fragments are obtained, the obtained fragment is Brazil banana, and if three fragments are obtained, the obtained fragment is Zhonghong No. 1 banana.
2. The CAPS marking method according to claim 1, wherein in step (3), Brazilian banana is obtained if two fragments with sizes of 447bp and 196bp, respectively, and Zhonghong No. 1 banana is obtained if three fragments with sizes of 447bp, 129bp and 77bp, respectively.
3. A primer pair for CAPS labeling method for distinguishing Brazil bananas and Banana Zhonghui No. 1, which is characterized in that:
forward primer G0629F 2: 5'-TAGCAAAATACATGAACTTTTGGA-3' the flow of the air in the air conditioner,
reverse primer G0629R 2: 5'-TCCTCACGACAACAACGAGAAAAAG-3' are provided.
4. An InDel molecular marker for distinguishing Brazil banana and Banana Zhonghong No. 1 is characterized in that the InDel molecular marker is positioned at the position of 128-138bp of a sequence shown by SEQ ID NO. 2, the sequence is CATCATTCTTG, the Banana Zhonghong No. 1 is an insertion of CATCATTCTTG, and the Brazil banana is a deletion of CATCATTCTTG.
5. Use of a reagent for detecting the InDel molecular marker of claim 4 for distinguishing Brazil banana from Zhonghong No. 1 banana.
6. Use of a primer pair that recognizes the InDel molecular marker of claim 4 in the preparation of a reagent that distinguishes Banana from Banana of Zhonghong No. 1.
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